The spermatozoan plasma and acrosomal membranes are partitioned into domains of distinct molecular and structural composition which display specific functions at defined fertilization steps. The long range goals of this proposal are to define the molecular and structural mechanisms which generate the mosaic character of the spermatozoan membranes and to identify the role of cytoskeletal assemblies in sperm development and fertilization. First we will determine if cytoskeletal assemblies function in the segregation of membrane proteins to selected domains by identifying plasma membrane components associated with the cytoskeleton and preparing antibodies against them to immunolocalize their surface distributions. The submembranous assembly with which they associate and changes in their cytoskeletal associations during sperm development and fertilization events will be determined the using electron microscopy and immunolocalization techniques. Second we will define the molecular and structural properties of the sperm membrane skeleton obtained from plasma membranes treated with nonionic detergents; we will employ electron microscopy to define its ultrastructure and electrophoretic techniques to define its polypeptide composition and specific protein-protein interactions with integral membrane proteins. Third we will isolate the acrosomal cytoskeleton utilizing cell fractionation procedures and define its polypeptide composition and interaction with specific components of the plasma membrane. Fourth we will characterize the detergent-stable aerosomal matrix components with respect to their polypeptide composition and define their role in segregating acrosomal hydrolases within the acrosome. Finally we will define the protein composition of a cytoskeletal assembly restricted to the sperm midpiece and determine if it functions in directing the mitochondria to the forming midpiece during spermiogenesis or in maintaining mitochondrial distribution in mature spermatozoa. Completion of these specific aims will provide new data on the mechanisms underlying the mosaic construction of the sperm plasma membrane and provide needed data on the molecular basis of specific fertilization events.